Gas Chromatography Separation Analysis Steps

The advantage of gas chromatography is that it can separate and analyze mixtures of multiple components. However, because there are many substances that can be used for chromatographic analysis, the appearance times of chromatographic peaks of different components on the same stationary phase may be the same, so it is difficult to characterize unknown substances based on chromatographic peaks alone. For an unknown sample, we must first understand its source, nature, and analytical purpose; on this basis, we can make a preliminary estimate of the sample; and then use a certain method to conduct qualitative identification based on known pure substances or relevant chromatographic qualitative reference data. Please see the following:

1. Source and pretreatment method of samples

Samples that can be directly analyzed by GC are usually gases or liquids. Solid samples should be dissolved in an appropriate solvent before analysis, and ensure that the samples do not contain components (such as inorganic salts) that cannot be analyzed by GC, which may damage the components of the chromatographic column. In this way, when we receive an unknown sample, we must understand the source, so as to estimate the components that the sample may contain and the boiling point range of the sample. If the sample system is simple and the sample components can be vaporized, it can be analyzed directly. If there are components in the sample that cannot be directly analyzed by GC, or the sample concentration is too low, necessary pretreatment must be carried out, such as adsorption, analysis, extraction, concentration, dilution, purification, derivatization and other methods to process the sample.

2. Determine the instrument configuration

The so-called instrument configuration refers to what sample injection device, what carrier gas, what chromatographic column and what detector are used to analyze samples.

Generally, the detector type should be determined first. FID detectors are often chosen for hydrocarbons, and ECD detectors are easy to choose for substances containing more electronegative groups (F, Cl, etc.) and less hydrocarbon content; when detection sensitivity requirements are not high or non-hydrocarbon components are included, TCD detectors can be chosen; for samples containing sulfur and phosphorus, FPD detectors can be chosen.

For liquid samples, you can choose the diaphragm pad injection method, and gas samples can use a six-way valve or adsorption thermal desorption injection method. General chromatography only configures the diaphragm pad injection method, so gas samples can be analyzed using adsorption-solvent analysis-diaphragm pad injection method.

Select suitable chromatographic columns according to the properties of the components to be tested, and generally follow the rule of similarity and compatibility. Select a non-polar column when separating non-polar substances, and select a polar column when separating polar substances. After the chromatographic column is determined, the working temperature of the chromatographic column is determined according to the difference in the distribution coefficients of the components to be tested in the sample. The isothermal method is adopted for simple systems, and the programmed temperature method is adopted for analysis for complex systems with large differences in distribution coefficients.

Commonly used carrier gases are hydrogen, nitrogen, helium, etc. Hydrogen and helium have small molecular weights and are often used as carrier gases for packed column chromatography; nitrogen has a large molecular weight and is often used as carrier gas for capillary gas chromatography; helium is used as carrier gas for gas chromatography mass spectrometry.

3. Determine initial operating conditions

When the sample is ready and the instrument configuration is determined, trial separation can begin. At this time, the initial separation conditions must be determined, which mainly include the injection volume, injection port temperature, detector temperature, column temperature and carrier gas flow rate. The injection volume is determined based on the sample concentration, column capacity and detector sensitivity. When the sample concentration does not exceed 10mg/mL, the injection volume of the packed column is usually 1- 5uL, while for a capillary column, if the split ratio is 50:1, the injection volume generally does not exceed 2uL. The temperature of the injection port is mainly determined by the boiling point range of the sample, and the operating temperature of the chromatographic column must also be considered. In principle, it is advantageous to have a higher temperature at the injection port, generally close to the boiling point of the component with the highest boiling point in the sample, but lower than the easily decomposed temperature.

4. Optimization of separation conditions

The purpose of optimization of separation conditions is to achieve the required separation results in the shortest analysis time. When the purpose of baseline separation cannot be achieved by changing the column temperature and the carrier gas flow rate, a longer chromatographic column should be replaced, or even a chromatographic column with a different stationary phase, because in GC, the chromatographic column is the key to the success of separation.

5. Qualitative identification

The so-called qualitative identification is to determine the attribution of chromatographic peaks. For simple samples, they can be characterized by reference materials. That is, under the same chromatographic conditions, inject the standard sample and the actual sample separately, and determine which peak on the chromatogram is the component to be analyzed according to the retention value. It must be noted that different compounds may have the same retention value on the same column, so it is not enough to use only one retention data for the qualitative determination of unknown samples. Double-column or multi-column retention index qualitative method is more reliable in GC, because the probability of different compounds having the same retention value on different columns is much smaller. Gas chromatography-mass spectrometry can be used when conditions permit.

6. Quantitative analysis

It is necessary to determine what quantitative method to use to determine the content of the component to be tested. Commonly used chromatographic quantitative methods are nothing more than peak area (peak height) percentage method, normalization method, internal standard method, external standard method and standard addition method (also called superposition method). The peak area (peak height) percentage method is the simplest but the least accurate. The method is optional only if the sample consists of homologues or is only for rough quantification. In comparison, the internal standard method has the highest quantitative accuracy because it quantifies using the response value relative to the standard (called the internal standard), which is added to the standard sample and the unknown sample respectively, so that errors due to fluctuations in operating conditions (including injection volume) can be offset. As for the standard addition method, a standard of the substance to be tested is quantitatively added to an unknown sample, and then a quantitative calculation is performed based on the increase in peak area (or peak height). The sample preparation process is similar to the internal standard method, but the calculation principle is entirely derived from the external standard method. The accuracy of standard addition legal quantity should be between the internal standard method and the external standard method.

7. Method verification

The so-called method verification is to prove the practicality and reliability of the developed method. Practicality generally refers to whether all the instrument configurations used can be purchased as commodities, whether the sample processing method is simple and easy to operate, whether the analysis time is reasonable, and whether the analysis cost is acceptable to peers. Reliability includes the linear range of quantification, limit of detection, method recovery, repeatability, reproducibility and accuracy.